This invention relates to transition metal complexes with at least one bidentate ligand containing at least one imidazole ring. In addition, the invention relates to the preparation of the transition metal complexes and to the use of the transition metal complexes as redox mediators.
Enzyme based electrochemical sensors are widely used in the detection of analytes in clinical, environmental, agricultural and biotechnological applications. Analytes that can be measured in clinical assays of fluids of the human body include, for example, glucose, lactate, cholesterol, bilirubin and amino acids. Levels of these analytes in biological fluids, such as blood, are important for the diagnosis and the monitoring of diseases.
Electrochemical assays are typically performed in cells with two or three electrodes, including at least one measuring or working electrode and one reference electrode. In three electrode systems, the third electrode is a counter-electrode. In two electrode systems, the reference electrode also serves as the counter-electrode. The electrodes are connected through a circuit, such as a potentiostat. The measuring or working electrode is a non-corroding carbon or metal conductor. Upon passage of a current through the working electrode, a redox enzyme is electrooxidized or electroreduced., The enzyme is specific to the analyte to be detected, or to a product of the analyte. The turnover rate of the enzyme is typically related (preferably, but not necessarily, linearly) to the concentration of the analyte itself, or to its product, in the test solution.
The electrooxidation or electroreduction of the enzyme is often facilitated by the presence of a redox mediator in the solution or on the electrode. The redox mediator assists in the electrical communication between the working electrode and the enzyme. The redox mediator can be dissolved in the fluid to be analyzed, which is in electrolytic contact with the electrodes, or can be applied within a coating on the working electrode in electrolytic contact with the analyzed solution. The coating is preferably not soluble in water, though it may swell in water. Useful devices can be made, for example, by coating an electrode with a film that includes a redox mediator and an enzyme where the enzyme is catalytically specific to the desired analyte, or its product. In contrast to a coated redox mediator, a diffusional redox mediator, which can be soluble or insoluble in water, functions by shuttling electrons between, for example, the enzyme and the electrode. In any case, when the substrate of the enzyme is electrooxidized, the redox mediator transports electrons from the substrate-reduced enzyme to the electrode; when the substrate is electroreduced, the redox mediator transports electrons from the electrode to the substrate-oxidized enzyme.
Recent enzyme based electrochemical sensors have employed a number of different redox mediators such as monomeric ferrocenes, quinoid-compounds including quinines (e.g., benzoquinones), nickel cyclamates, and ruthenium ammines. For the most part, these redox mediators have one or more of the following limitations: the solubility of the redox mediators in the test solutions is low, their chemical, light, thermal, or pH stability is poor, or they do not exchange electrons rapidly enough with the enzyme or the electrode or both. Additionally, the redox potentials of many of these reported redox mediators are so oxidizing that at the potential where the reduced mediator is electrooxidized on the electrode, solution components other than the analyte are also electrooxidized; in other cases they are so reducing that solution components, such as, for example, dissolved oxygen are also rapidly electroreduced. As a result, the sensor utilizing the mediator is not sufficiently specific.
The present invention is directed to novel transition metal complexes. The present invention is also directed to the use of the complexes as redox mediators. The preferred redox mediators typically exchange electrons rapidly with enzymes and electrodes, are stable, and have a redox potential that is tailored for the electrooxidation of analytes, exemplified by glucose.
One embodiment of the invention is a transition metal complex having the formula: 
M is cobalt, ruthenium, osmium, or vanadium. L is selected from the group consisting of: 
R1, R2, and Rxe2x80x21 are independently substituted or unsubstituted alkyl, alkenyl, or aryl groups. R3, R4, R5, R6, Rxe2x80x23, Rxe2x80x24, Ra, Rb, Rc, and Rd are independently xe2x80x94H, xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, xe2x80x94NO2, xe2x80x94CN, xe2x80x94CO2H, xe2x80x94SO3H, xe2x80x94NHNH2, xe2x80x94SH, aryl, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, xe2x80x94OH, alkoxy, xe2x80x94NH2, alkylamino, dialkylamino, alkanoylamino, arylcarboxamido, hydrazino, alkylhydrazino, hydroxylamino, alkoxyamino, alkylthio, alkenyl, aryl, or alkyl. c is an integer selected from xe2x88x921 to xe2x88x925 or +1 to +5 indicating a positive or negative charge. X represents at least one counter ion and d is an integer from 1 to 5 representing the number of counter ions, X. L1, L2, L3 and L4 are other ligands.
Another embodiment is a redox mediator having the formula: 
M is iron, cobalt, ruthenium, osmium, or vanadium. L is a bidentate ligand comprising at least one imidazole ring. c is an integer selected from xe2x88x921 to xe2x88x925 or +1 to +5 indicating a positive or negative charge. X represents at least one counter ion and d is an integer from 1 to 5 representing the number of counter ions, X. L1, L2, L3 and L4 are other ligands.
Another embodiment is a sensor that includes the redox polymer, a working electrode, and a counter electrode. The redox polymer is disposed proximate to the working electrode.
Yet another embodiment is a polymer that includes a polymeric backbone and a transition metal complex having the following formula: 
M is iron, cobalt, ruthenium, osmium, or vanadium. L is a bidentate ligand comprising at least one imidazole ring. c is an integer selected from xe2x88x921 to xe2x88x925 or +1 to +5 indicating a positive or negative charge. X represents at least one counter ion and d is an integer from 1 to 5 representing the number of counter ions, X. L1, L2, L3 and L4 are other ligands where at least one of L, L1, L2, L3 and L4 couples to the polymeric backbone.
When used herein, the following definitions define the stated term:
The term xe2x80x9calkylxe2x80x9d includes linear or branched, saturated aliphatic hydrocarbons. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl and the like. Unless otherwise noted, the term xe2x80x9calkylxe2x80x9d includes both alkyl and cycloalkyl groups.
The term xe2x80x9calkoxyxe2x80x9d describes an alkyl group joined to the remainder of the structure by an oxygen atom. Examples of alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, butoxy, tert-butoxy, and the like. In addition, unless otherwise noted, the term xe2x80x98alkoxyxe2x80x99 includes both alkoxy and cycloalkoxy groups.
The term xe2x80x9calkenylxe2x80x9d describes an unsaturated, linear or branched aliphatic hydrocarbon having at least one carbon-carbon double bond. Examples of alkenyl groups include ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-methyl-1-propenyl, and the like.
A xe2x80x9creactive groupxe2x80x9d is a functional group of a molecule that is capable of reacting with another compound to couple at least a portion of that other compound to the molecule. Reactive groups include carboxy, activated ester, sulfonyl halide, sulfonate ester, isocyanate, isothiocyanate, epoxide, aziridine, halide, aldehyde, ketone, amine, acrylamide, thiol, acyl azide, acyl halide, hydrazine, hydroxylamine, alkyl halide, imidazole, pyridine, phenol, alkyl sulfonate, halotriazine, imido ester, maleimide, hydrazide, hydroxy, and photo-reactive azido aryl groups. Activated esters, as understood in the art, generally include esters of succinimidyl, benzotriazolyl, or aryl substituted by electron-withdrawing groups such as sulfo, nitro, cyano, or halo groups; or carboxylic acids activated by carbodiimides.
A xe2x80x9csubstitutedxe2x80x9d functional group (e.g., substituted alkyl, alkenyl, or alkoxy group) includes at least one substituent selected from the following: halogen, alkoxy, mercapto, aryl, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, xe2x80x94OH, xe2x80x94NH2, alkylamino, dialkylamino, trialkylammonium, alkanoylamino, arylcarboxamido, hydrazino, alkylthio, alkenyl, and reactive groups.
A xe2x80x9cbiological fluidxe2x80x9d is any body fluid or body fluid derivative in which the analyte can be measured, for example, blood, interstitial fluid, plasma, dermal fluid, sweat, and tears.
An xe2x80x9celectrochemical sensorxe2x80x9d is a device configured to detect the presence of or measure the concentration or amount of an analyte in a sample via electrochemical oxidation or reduction reactions. These reactions typically can be transduced to an electrical signal that can be correlated to an amount or concentration of analyte.
A xe2x80x9credox mediatorxe2x80x9d is an electron transfer agent for carrying electrons between an analyte or an analyte-reduced or analyte-oxidized enzyme and an electrode, either directly, or via one or more additional electron transfer agents.
xe2x80x9cElectrolysisxe2x80x9d is the electrooxidation or electroreduction of a compound either directly at an electrode or via one or more electron transfer agents (e.g., redox mediators or enzymes).
The term xe2x80x9creference electrodexe2x80x9d includes both a) reference electrodes and b) reference electrodes that also function as counter electrodes (i.e., counter/reference electrodes), unless otherwise indicated.
The term xe2x80x9ccounter electrodexe2x80x9d includes both a) counter electrodes and b) counter electrodes that also function as reference electrodes (i.e., counter/reference electrodes), unless otherwise indicated.
Generally, the present invention relates to transition metal complexes of iron, cobalt, ruthenium, osmium, and vanadium having at least one bidentate ligand containing an imidazole ring. The invention also relates to the preparation of the transition metal complexes and to the use of the transition metal complexes as redox mediators. In at least some instances, the transition metal complexes have one or more of the following characteristics: redox potentials in a particular range, the ability to exchange electrons rapidly with electrodes, the ability to rapidly transfer electrons to or rapidly accept electrons from an enzyme to accelerate the kinetics of electrooxidation or electroreduction of an analyte in the presence of an enzyme or another analyte-specific redox catalyst. For example, a redox mediator may accelerate the electrooxidation of glucose in the presence of glucose oxidase or PQQ-glucose dehydrogenase, a process that can be useful for the selective assay of glucose in the presence of other electrochemically oxidizable species. Compounds having the formula 1 are examples of transition metal complexes of the present invention. 
M is a transition metal and is typically iron, cobalt, ruthenium, osmium, or vanadium. Ruthenium and osmium are particularly suitable for redox mediators.
L is a bidentate ligand containing at least one imidazole ring. One example of L is a 2,2xe2x80x2-biimidazole having the following structure 2: 
R1 and R2 are substituents attached to two of the 2,2xe2x80x2-biimidazole nitrogens and are independently substituted or unsubstituted alkyl, alkenyl, or aryl groups. Generally, R1 and R2 are unsubstituted C1 to C12 alkyls. Typically, R1 and R2 are unsubstituted C1 to C4 alkyls. In some embodiments, both R1 and R2 are methyl.
R3, R4, R5, and R6 are substituents attached to carbon atoms of the 2,2xe2x80x2-biimidazole and are independently xe2x80x94H, xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, xe2x80x94NO2, xe2x80x94CN, xe2x80x94CO2H, xe2x80x94SO3H, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, xe2x80x94OH, alkoxy, xe2x80x94NH2, alkylamino, dialkylamino, alkanoylamino, arylcarboxamido, hydrazino, alkylhydrazino, hydroxylamino, alkoxyamino, alkylthio, alkenyl, aryl, or alkyl. Alternatively, R3 and R4 in combination or R5 and R6 in combination independently form a saturated or unsaturated 5- or 6-membered ring. An example of this is a 2,2xe2x80x2-bibenzoimidazole derivative. Typically, the alkyl and alkoxy portions are C1 to C12. The alkyl or aryl portions of any of the substituents are optionally substituted by xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, alkylamino, dialkylamino, trialkylammonium (except on aryl portions), alkoxy, alkylthio, aryl, or a reactive group. Generally, R3, R4, R5, and R6 are independently xe2x80x94H or unsubstituted alkyl groups. Typically, R3, R4, R5, and R6 are xe2x80x94H or unsubstituted C1 to C12 alkyls. In some embodiments, R3, R4, R5, and R6 are all xe2x80x94H.
Another example of L is a 2-(2-pyridyl)imidazole having the following structure 3: 
Rxe2x80x21 is a substituted or unsubstituted aryl, alkenyl, or alkyl. Generally, Rxe2x80x21 is a substituted or unsubstituted C1-C12 alkyl. Rxe2x80x21 is typically methyl or a C1-C12 alkyl that is optionally substituted with a reactive group.
Rxe2x80x23, Rxe2x80x24, Ra, Rb, Rc, and Rd are independently xe2x80x94H, xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, xe2x80x94NO2, xe2x80x94CN, xe2x80x94CO2H, xe2x80x94SO3H, xe2x80x94NHNH2, xe2x80x94SH, alkoxylcarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, xe2x80x94OH, alkoxy, xe2x80x94NH2, alkylamino, dialkylamino, alkanoylamino, arylcarboxamido, hydrazino, alkylhydrazino, hydroxylamino, alkoxylamino, alkylthio, alkenyl, aryl, or alkyl. Alternatively, Rc and Rd in combination or Rxe2x80x23 and Rxe2x80x24 in combination can form a saturated or unsaturated 5- or 6-membered ring. Typically, the alkyl and alkoxy portions are C1 to C12. The alkyl or aryl portions of any of the substituents are optionally substituted by xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, alkylamino, dialkylamino, trialkylammonium (except on aryl portions), alkoxy, alkylthio, aryl, or a reactive group. Generally, Rxe2x80x23, Rxe2x80x24, Ra, Rb, Rc and Rd are independently xe2x80x94H or unsubstituted alkyl groups. Typically, Ra and Rc are xe2x80x94H and Rxe2x80x23, Rxe2x80x24, Rb, and Rd are xe2x80x94H or methyl.
c is an integer indicating the charge of the complex. Generally, c is an integer selected from xe2x88x921 to xe2x88x925 or +1 to +5 indicating a positive or negative charge. For a number of osmium complexes, c is +2 or +3.
X represents counter ion(s). Examples of suitable counter ions include anions, such as halide (e.g., fluoride, chloride, bromide or iodide), sulfate, phosphate, hexafluorophosphate, and tetrafluoroborate, and cations (preferably, monovalent cations), such as lithium, sodium, potassium, tetralkylammonium, and ammonium. Preferably, X is a halide, such as chloride. The counter ions represented by X are not necessarily all the same.
d represents the number of counter ions and is typically from 1 to 5.
L1, L2, L3 and L4 are ligands attached to the transition metal via a coordinative bond. L1, L2, L3 and L4 can be monodentate ligands or, in any combination, bi-, ter-, or tetradentate ligands For example, L1, L2, L3 and L4 can combine to form two bidentate ligands such as, for example, two ligands selected from the group of substituted and unsubstituted 2,2xe2x80x2-biimidazoles, 2-(2-pyridyl)imidizoles, and 2,2xe2x80x2-bipyridines
Examples of other L1, L2, L3 and L4 combinations of the transition metal complex include:
(A) L1 is a monodentate ligand and L2, L3 and L4 in combination form a terdentate ligand;
(B) L1 and L2 in combination are a bidentate ligand, and L3 and L4 are the same or different monodentate ligands;
(C) L1 and L1, in combination, and L3 and L4 in combination form two independent bidentate ligands which can be the same or different; and
(D) L1, L2, L3 and L4 in combination form a tetradentate ligand.
Examples of suitable monodentate ligands include, but are not limited to, xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, xe2x80x94CN, xe2x80x94SCN, xe2x80x94OH, H2O, NH3, alkylamine, dialkylamine, trialkylamine, alkoxy or heterocyclic compounds. The alkyl or aryl portions of any of the ligands are optionally substituted by xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, alkylamino, dialkylamino, trialkylammonium (except on aryl portions), alkoxy, alkylthio, aryl, or a reactive group. Any alkyl portions of the monodentate ligands generally contain 1 to 12 carbons. More typically, the alkyl portions contain 1 to 6 carbons. In other embodiments, the monodentate ligands are heterocyclic compounds containing at least one nitrogen, oxygen, or sulfur atom. Examples of suitable heterocyclic monodentate ligands include imidazole, pyrazole, oxazole, thiazole, pyridine, pyrazine and derivatives thereof. Suitable heterocyclic monodentate ligands include substituted and unsubstituted imidazole and substituted and unsubstituted pyridine having the following general formulas 4 and 5, respectively: 
With regard to formula 4, R7 is generally a substituted or unsubstituted alkyl, alkenyl, or aryl group. Typically, R7 is a substituted or unsubstituted C1 to C12 alkyl or alkenyl. The substitution of inner coordination sphere chloride anions by imidazoles does not typically cause a large shift in the redox potential in the oxidizing direction, differing in this respect from substitution by pyridines, which typically results in a large shift in the redox potential in the oxidizing direction.
R8, R9 and R10 are independently xe2x80x94H, xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, xe2x80x94NO2, xe2x80x94CN, xe2x80x94CO2H, xe2x80x94SO3H, xe2x80x94NHNH2, xe2x80x94SH, aryl, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, xe2x80x94OH, alkoxy, xe2x80x94NH2, alkylamino, dialkylamino, alkanoylamino, arylcarboxamido, hydrazino, alkylhydrazino, hydroxylamino, alkoxyamino, alkylthio, alkenyl, aryl, or alkyl. Alternatively, R9 and R10, in combination, form a fused 5 or 6-membered ring that is saturated or unsaturated. The alkyl portions of the substituents generally contain 1 to 12 carbons and typically contain 1 to 6 carbon atoms. The alkyl or aryl portions of any of the substituents are optionally substituted by xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, alkylamino, dialkylamino, trialkylammonium (except on aryl portions), alkoxy, alkylthio, aryl, or a reactive group. In some embodiments, R8, R9 and R10 are xe2x80x94H or substituted or unsubstituted alkyl. Preferably, R8, R9 and R10 are xe2x80x94H.
With regard to Formula 5, R11, R12, R13, R14 and R15 are independently xe2x80x94H, xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, xe2x80x94NO2, xe2x80x94CN, xe2x80x94CO2H, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, xe2x80x94OH, alkoxy, xe2x80x94NH2, alkylamino, dialkylamino, alkanoylamino, arylcarboxamido, hydrazino, alkylhydrazino, hydroxylamino, alkoxyamino, alkylthio, alkenyl, aryl, or alkyl. The alkyl or aryl portions of any of the substituents are optionally substituted by xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, alkylamino, dialkylamino, trialkylammonium (except for aryl portions), alkoxy, alkylthio, aryl, or a reactive group. Generally, R11, R12, R13, R14 and R15 are xe2x80x94H, methyl, C1-C2 alkoxy, C1-C2 alkylamino, C2-C4 dialkylamino, or a C1-C6 lower alkyl substituted with a reactive group.
One example includes R11 and R15 as xe2x80x94H, R12 and R14 as the same and xe2x80x94H or methyl, and R13 as xe2x80x94H, C1 to C12 alkoxy, xe2x80x94NH2, C1 to C12 alkylamino, C2 to C24 dialkylamino, hydrazino, C1 to C12 alkylhydrazino, hydroxylamino, C1 to C12 alkoxyamino, C1 to C12 alkylthio, or C1 to C12 alkyl. The alkyl or aryl portions of any of the substituents are optionally substituted by xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, alkylamino, dialkylamino, trialkylammonium (except on aryl portions), alkoxy, alkylthio, aryl, or a reactive group.
Examples of suitable bidentate ligands include, but are not limited to, amino acids, oxalic acid, acetylacetone, diaminoalkanes, ortho-diaminoarenes, 2,2xe2x80x2-biimidazole, 2,2xe2x80x2-bioxazole, 2,2xe2x80x2-bithiazole, 2-(2-pyridyl)imidazole, and 2,2xe2x80x2-bipyridine and derivatives thereof. Particularly suitable bidentate ligands for redox mediators include substituted and unsubstituted 2,2xe2x80x2-biimidazole, 2-(2-pyridyl)imidazole and 2,2xe2x80x2-bipyridine. The substituted 2,2xe2x80x2 biimidazole and 2-(2-pyridyl)imidazole ligands can have the same substitution patterns described above for the other 2,2xe2x80x2-biimidazole and 2-(2-pyridyl)imidazole ligand. A 2,2xe2x80x2-bipyridine ligand has the following general formula 6: 
R16, R17, R18, R19, R20, R21, R22 and R23 are independently xe2x80x94H, xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94CN, xe2x80x94CO2H, xe2x80x94SO3H, xe2x80x94NHNH2, xe2x80x94SH, aryl, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, xe2x80x94OH, alkoxy, xe2x80x94NH, alkylamino, dialkylamino, alkanoylamino, arylcarboxamido, hydrazino, alkylhydrazino, hydroxylamino, alkoxylamino, alkylthio, alkenyl, or alkyl. Typically, the alkyl and alkoxy portions are C1 to C12. The alkyl or aryl portions of any of the substituents are optionally substituted by xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, alkylamino, dialkylamino, trialkylammonium (except on aryl portions), alkoxy, alkylthio, aryl, or a reactive group.
Specific examples of suitable combinations of R16, R17, R18, R19, R20, R21, R22 and R23 include R16 and R23 as H or methyl; R17 and R22 as the same and xe2x80x94H or methyl; and R19 and R20 as the same and xe2x80x94H or methyl. An alternative combination is where one or more adjacent pairs of substituents R16 and R17, on the one hand, and R22 and R23, on the other hand, independently form a saturated or unsaturated 5- or 6-membered ring. Another combination includes R19 and R20 forming a saturated or unsaturated five or six membered ring.
Another combination includes R16, R17, R19, R20, R22 and R23 as the same and xe2x80x94H and R18 and R21 as independently xe2x80x94H, alkoxy, xe2x80x94NH2, alkylamino, dialkylamino, alkylthio, alkenyl, or alkyl. The alkyl or aryl portions of any of the substituents are optionally substituted by xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, alkylamino, dialkylamino, trialkylammonium (except on aryl portions), alkoxy, alkylthio, aryl, or a reactive group. As an example, R18 and R21 can be the same or different and are xe2x80x94H, C1-C6 alkyl, C1-C6 amino, C1 to C12 alkylamino, C2 to C12 dialkylamino, C1 to C12 alkylthio, or C1 to C12 alkoxy, the alkyl portions of any of the substituents are optionally substituted by a xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, aryl, C2 to C12 dialkylamino, C3 to C18 trialkylammonium, C1 to C6 alkoxy, C1 to C6 alkylthio or a reactive group.
Examples of suitable terdentate ligands include, but are not limited to, diethylenetriamine, 2,2xe2x80x2,2xe2x80x3-terpyridine, 2,6-bis(N-pyrazolyl)pyridine, and derivatives of these compounds. 2,2xe2x80x2,2xe2x80x3-terpyridine and 2,6-bis(N-pyrazolyl)pyridine have the following general formulas 7 and 8 respectively: 
With regard to formula 7, R24, R25 and R26 are independently xe2x80x94H or substituted or unsubstituted C1 to C12 alkyl. Typically, R24, R25 and R26 are xe2x80x94H or methyl and, in some embodiments, R24 and R26 are the same and are xe2x80x94H. Other substituents at these or other positions of the compounds of formulas 7 and 8 can be added.
With regard to formula 8, R27, R28 and R29 are independently xe2x80x94H, xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, xe2x80x94NO2, xe2x80x94CN, xe2x80x94CO2H, xe2x80x94SO3H, xe2x80x94NHNH2, xe2x80x94SH, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, xe2x80x94OH, alkoxy, xe2x80x94NH, alkylamino, dialkylamino, alkanoylamino, arylcarboxamido, hydrazino, alkylhydrazino, hydroxylamino, alkoxylamino, alkylthio, alkenyl, aryl, or alkyl. The alkyl or aryl portions of any of the substituents are optionally substituted by xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, alkylamino, dialkylamino, trialkylammonium (except on aryl portions), alkoxy, alkylthio, aryl, or a reactive group. Typically, the alkyl and alkoxy groups are C1 to C12 and, in some embodiments, R27 and R29 are the same and are xe2x80x94H.
Examples of suitable tetradentate ligands include, but are not limited to, triethylenetriamine, ethylenediaminediacetic acid, tetraaza macrocycles and similar compounds as well as derivatives thereof.
Examples of suitable transition metal complexes are illustrated using Formula 9 and 10: 
With regard to transition metal complexes of formula 9, the metal osmium is complexed to two substituted 2,2xe2x80x2-biimidazole ligands and one substituted or unsubstituted 2,2xe2x80x2-bipyridine ligand. R1, R2, R3, R4, R5, R6, R16, R17, R18, R19, R20, R21, R22, R23, c, d, and X are the same as described above.
In one embodiment, R1 and R2 are methyl; R3, R4, R5, R6, R16, R17, R19, R20, R21, R22, and R23 are xe2x80x94H; and R18 and R21 are the same and are xe2x80x94H, methyl, or methoxy. Preferably, R18 and R21 are methyl or methoxy.
In another embodiment, R1 and R2 are methyl; R3, R4, R5, R6, R16, R17, R18, R19, R20, R22 and R23 are xe2x80x94H; and R21 is halo, C1 to C12 alkoxy, C1 to C12 alkylamino, or C2 to C24 dialkylamino. The alkyl or aryl portions of any of the substituents are optionally substituted by xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, alkylamino, dialkylamino, trialkylammonium (except on aryl portions), alkoxy, alkylthio, aryl, or a reactive group. For example, R21 is a C1 to C12 alkylamino or C2 to C24 dialkylamino, the alkyl portion(s) of which are substituted with a reactive group, such as a carboxylic acid, activated ester, or amine. Typically, the alkylamino group has 1 to 6 carbon atoms and the dialkylamino group has 2 to 8 carbon atoms.
With regard to transition metal complexes of formula 10, the metal osmium is complexed to two substituted 2,2xe2x80x2-biimidazole ligands and one substituted or unsubstituted 2-(2-pyridyl)imidazole ligand. R1, R2, R3, R4, R5, Rxe2x80x21, Rxe2x80x22, Rxe2x80x23, Rxe2x80x24, Ra, Rb, Rc, Rd, c, d, and X are the same as described above.
In one embodiment, R1 and R2are methyl; R3, R4, R5, R6, Rxe2x80x23, Rxe2x80x24 and Rd are independently xe2x80x94H or methyl; Ra and Rc are the same and are xe2x80x94H; and Rb is C1 to C12 alkoxy, C1 to C12 alkylamino, or C2 to C24 dialkylamino. The alkyl or aryl portions of any of the substituents are optionally substituted by xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, alkylamino, dialkylamino, trialkylammonium (except on aryl portions), alkoxy, alkylthio, aryl, or a reactive group.
A list of specific examples of preferred transition metal complexes with respective redox potentials is shown in Table 1.
The transition metal complexes of Formula 1 also include transition metal complexes that are coupled to a polymeric backbone through one or more of L, L1, L2, L3, and L4. Additional examples of suitable transition metal complexes are described in U.S. patent application Ser. No. 09/712,065 allowed, entitled xe2x80x9cPolymeric Transition Metal Complexes and Uses Thereofxe2x80x9d, filed on even date herewith, incorporated herein by reference. In some embodiments, the polymeric backbone has functional groups that act as ligands of the transitional metal complex. Such polymeric backbones include, for example, poly(4-vinylpyridine) and poly(N-vinylimidazole) in which the pyridine and imidazole groups, respectively, can act as monodentate ligands of the transition metal complex. In other embodiments, the transition metal complex can be the reaction product between a reactive group on a precursor polymer and a reactive group on a ligand of a precursor transition metal complex (such as a complex of Formula 1 where one of L, L1, L2, L3 and L4 includes a reactive group as described above). Suitable precursor polymers include, for example, poly(acrylic acid) Formula 11), styrene/maleic anhydride copolymer (Formula 12), methylvinylether/maleic anhydride copolymer (GANTEX polymer) (Formula 13), poly(vinylbenzylchloride) (Formula 14), poly(allylamine) (Formula 15), polylysine (Formula 16), carboxy-poly(vinylpyridine (Formula 17), and poly(sodium 4-styrene sulfonate) (Formula 18). 
Alternatively, the transition metal complex can have reactive group(s) for immobilization or conjugation of the complexes to other substrates or carriers, examples of which include, but are not limited to, macromolecules (e.g., enzymes) and surfaces (e.g., electrode surfaces).
For reactive attachment to polymers, substrates, or other carriers, the transition metal complex precursor includes at least one reactive group that reacts with a reactive group on the polymer, substrate, or carrier. Typically, covalent bonds are formed between the two reactive groups to generate a linkage. Examples of such linkages are provided in Table 2, below. Generally, one of the reactive groups is an electrophile and the other reactive group is a nucleophile.
Transition metal complexes of the present invention can be soluble in water or other aqueous solutions, or in organic solvents. In general, the transition metal complexes can be made soluble in either aqueous or organic solvents by having an appropriate counter ion or ions, X. For example, transition metal complexes with small counter anions, such as Fxe2x88x92, Clxe2x88x92, and Brxe2x88x92, tend to be water soluble. On the other hand, transition metal complexes with bulky counter anions, such as Ixe2x88x92, BF4xe2x88x92 and PF6xe2x88x92, tend to be soluble in organic solvents. Preferably, the solubility of transition metal complexes of the present invention is greater than about 0.1 M (moles/liter) at 25xc2x0 C. for a desired solvent.
The transition metal complexes discussed above are useful as redox mediators in electrochemical sensors for the detection of analytes in bio-fluids. The use of transition metal complexes as redox mediators is described, for example, in U.S. Pat. Nos. 5,262,035; 5,262,305; 5,320,725; 5,365,786; 5,593,852; 5,665,222; 5,972,199; and 6,143,164 and U.S. patent applications Ser. Nos. 09/034,372, (now U.S. Pat. No. 6,134,461); U.S. Ser. No. 09/070,677, (now pending U.S. Pat. No. 6,175,752); U.S. Ser. No. 09/295,962, (now U.S. Pat. No. 6,338,790) and U.S. Ser. No. 09/434,026, all of which are herein incorporated by reference. The transitional metal complexes described herein can typically be used in place of those discussed in the references listed above. The transitions metal complexes that include a polymeric backbone and are redox mediators can also be referred to as xe2x80x9credox polymersxe2x80x9d.
In general, the redox mediatoris-disposed on or in proximity to (e.g., in a solution surrounding) a working electrode. The redox mediator transfers electrons between the working electrode and an analyte. In some preferred embodiments, an enzyme is also included to facilitate the transfer. For example, the redox mediator transfers electrons between the working electrode and glucose (typically via an enzyme) in an enzyme-catalyzed reaction of glucose. Redox polymers are particularly useful for forming non-leachable coatings on the working electrode. These can be formed, for example, by crosslinking the redox polymer on the working electrode, or by crosslinking the redox polymer and the enzyme on the working electrode
Transition metal complexes can enable accurate, reproducible and quick or continuous assays. Transition metal complex redox mediators accept electrons from, or transfer electrons to, enzymes or analytes at a high rate and also exchange electrons rapidly with an electrode. Typically, the rate of self exchange, the process in which a reduced redox mediator transfers an electron to an oxidized redox mediator, is rapid. At a defined redox mediator concentration, this provides for more rapid transport of electrons between the enzyme (or analyte) and electrode, and thereby shortens the response time of the sensor. Additionally, the novel transition metal complex redox mediators are typically stable under ambient light and at the temperatures encountered in use, storage and transportation. Preferably, the transition metal complex redox mediators do not undergo chemical change, other than oxidation and reduction, in the period of use or under the conditions of storage, though the redox mediators can be designed to be activated by reacting, for example, with water or the analyte.
The transition metal complex can be used as a redox mediator in combination with a redox enzyme to electrooxidize or electroreduce the analyte or a compound derived of the analyte, for example by hydrolysis of the analyte. The redox potentials of the redox mediators are generally more positive (i.e. more oxidizing) than the redox potentials of the redox enzymes when the analyte is electrooxidized and more negative when the analyte is electroreduced. For example, the redox potentials of the preferred transition metal complex redox mediators used for electrooxidizing glucose with glucose oxidase or PQQ-glucose dehydrogenase as enzyme is between about xe2x88x92200 mV and +200 mV versus a Ag/AgCl reference electrode, and the most preferred mediators have redox potentials between about xe2x88x92100 mV and about +100 mV versus a Ag/AgCl reference electrode
Electron transport involves an exchange of electrons between segments of the redox polymers (e.g., one or more transition metal complexes coupled to a polymeric backbone, as described above) in a crosslinked film disposed on an electrode. The transition metal complex can be bound to the polymer backbone though covalent, coordinative or ionic bonds, where covalent and coordinative binding are preferred. Electron exchange occurs, for example, through the collision of different segments of the crosslinked redox polymer. Electrons transported through the redox polymer can originate from, for example, electrooxidation or electroreduction of an enzymatic substrate, such as, for example, the oxidation of glucose by glucose oxidase.
The degree of crosslinking of the redox polymer can influence the transport of electrons or ions and thereby:the rates of the electrochemical reactions. Excessive crosslinking of the polymer can reduce the mobility of the segments of the redox polymer. A reduction in segment mobility can slow the diffusion of electrons or ions through the redox polymer film. A reduction in the diffusivity of electrons, for example, can require a concomitant reduction in the thickness of the film on the electrode where electrons or electron vacancies are collected or delivered. The degree of crosslinking in a redox polymer film can thus affect the transport of electrons from, for example, an enzyme to the transition metal redox centers of the redox polymer such as, for example, Os2+/3+ metal redox centers; between redox centers of the redox polymer; and from these transition metal redox centers to the electrode.
Inadequate crosslinking of a redox polymer can result in excessive swelling of the redox polymer film and to the leaching of the components of the redox polymer film. Excessive swelling can also result in the migration of the swollen polymer into the analyzed solution, in the softening of the redox polymer film, in the film""s susceptibility to removal by shear, or any combination of these effects.
Crosslinking can decrease the leaching of film components and can improve the mechanical stability of the film under shear stress. For example, as disclosed in Binyamin, G. and Heller, A; Stabilization of Wired Glucose Oxidase Anodes Rotating at 1000 rpm at 37xc2x0 C.; Journal of the Electrochemical Society, 146(8), 2965-2967, 1999, herein incorporated by reference, replacing a difunctional crosslinker, such as polyethylene glycol diglycidyl ether, with a trifunctional crosslinker such as N,N-diglycidyl-4-glycidyloxyaniline, for example, can reduce leaching and shear problems associated with inadequate crosslinking.
Examples of other bifunctional, trifunctional and tetrafunctional crosslinkers are listed below:
Amine-reactive Bifunctional Crosslinkers 
Pyridine- or Imidazole-reactive Bifunctional Crosslinkers 
Pyridine- or Imidazole-reactive Trifunctional Crosslinker 
Pyridine- or Imidazole-reactive Tetrafunctional Crosslinkers 
Alternatively, the number of crosslinking sites can be increased by reducing the number of transition metal complexes attached to the polymeric backbone, thus making more polymer pendant groups available for crosslinking. One important advantage of at least some of the redox polymers is the increased mobility of the pendant transition metal complexes, resulting from the flexibility of the pendant groups. As a result, in at least some embodiments, fewer transition metal complexes per polymer backbone are needed to achieve a desired level of diffusivity of electrons and current density of analyte electrooxidation or electroreduction.
Transition metal complexes can be directly or indirectly attached to a polymeric backbone, depending on the availability and nature of the reactive groups on the complex and the polymeric backbone. For example, the pyridine groups in poly(4-vinylpyridine) or the imidazole groups in poly(N-vinylimidazole) are capable of acting as monodentate ligands and thus can be attached to a metal center directly. Alternatively, the pyridine groups in poly(4-vinylpyridine) or the imidazole groups in poly(N-vinylimidazole) can be quaternized with a substituted alkyl moiety having a suitable reactive group, such as a carboxylate function, that can be activated to form a covalent bond with a reactive group, such as an amine, of the transition metal complex. (See Table 2 for a list of other examples of reactive groups.)
Redox centers such as, for example, Os2+/3+ can be coordinated with five heterocyclic nitrogens and an additional ligand such as, for example, a chloride anion. An example of such a coordination complex includes two bipyridine ligands which form stable coordinative bonds, the pyridine of poly(4-vinylpyridine) which forms a weaker coordinative bond, and a chloride anion which forms the least stable coordinative bond.
Alternatively, redox centers, such as Os2+/3+, can be coordinated with six heterocyclic nitrogen atoms in its inner coordination sphere. The six coordinating atoms are preferably paired in the ligands, for example, each ligand is composed of at least two rings. Pairing of the coordinating atoms can influence the potential of an electrode used in conjunction with redox polymers of the present invention.
Typically, for analysis of glucose, the potential at which the working electrode, coated with the redox polymer, is poised is negative of about +250 mV vs. SCE (standard calomel electrode). Preferably, the electrode is poised negative of about +150 mV vs. SCE. Poising the electrode at these potentials reduces the interfering electrooxidation of constituents of biological solutions such as, for example, urate, ascorbate and acetaminophen. The potential can be modified by altering the ligand structure of the complex.
The redox potential of a redox polymer, as described herein, is related to the potential at which the electrode is poised. Selection of a redox polymer with a desired redox potential allows tuning of the potential at which the electrode is best poised. The redox potentials of a number of the redox polymers described herein are negative of about +150 mV vs. SCE and can be negative of about +50 mV vs. SCE to allow the poising of the electrode potentials negative of about +250 mV vs. SCE and preferably negative of about +150 mV vs. SCE.
The strength of the coordination bond can influence the potential of the redox centers in the redox polymers. Typically, the stronger the coordinative bond, the more positive the redox potential. A shift in the potential of a redox center resulting from a change in the coordination sphere of the transition metal can produce a labile transition metal complex. For example, when the redox potential of an Os2+/3+ complex is downshifted by changing the coordination sphere, the complex becomes labile. Such a labile transition metal complex may be undesirable when fashioning a metal complex polymer for use as a redox mediator and can be avoided through the use of weakly coordinating multidentate or chelating heterocyclics as ligands.
Transition metal complexes used as redox mediators in electrodes can be affected by the presence of transition metals in the analyzed sample including, for example, Fe3+ or Zn2+. The addition of a transition metal cation to a buffer used to test an electrode results in a decline in the current produced. The degree of current decline depends on the presence of anions in the buffer which precipitate the transition metal cations. The lesser the residual concentration of transition metal cations in the sample solution, the more stable the current. Anions which aid in the precipitation of transition metal cations include, for example, phosphate. It has been found that a decline in current upon the addition of transition metal cations is most pronounced in non-phosphate buffers. If an electrode is transferred from a buffer containing a transition metal cation to a buffer substantially free of the transition metal cation, the original current is restored.
The decline in current is thought to be due to additional crosslinking of a pyridine-containing polymer backbone produced by the transition metal cations. The transition metal cations can coordinate nitrogen atoms of different chains and chain segments of the polymers. Coordinative crosslinking of nitrogen atoms of different chain segments by transition metal cations can reduce the diffusivity of electrons.
Serum and other physiological fluids contain traces of transition metal ions, which can diffuse into the films of electrodes made with the redox polymers of the present invention, lowering the diffusivity of electrons and thereby the highest current reached at high analyte concentration. In addition, transition metal ions like iron and copper can bind to proteins of enzymes and to the reaction centers or channels of enzymes, reducing their turnover rate. The resulting decrease in sensitivity can be remedied through the use of anions which complex with interfering transition metal ions, for example, in a buffer employed during the production of the transition metal complex. A non-cyclic polyphosphate such as, for example, pyrophosphate or triphosphate, can be used. For example, sodium or potassium non-cyclic polyphosphate buffers can be used to exchange phosphate anions for those anions in the transition metal complex which do not precipitate transition metal ions. The use of linear phosphates can alleviate the decrease in sensitivity by forming strong complexes with the damaging transition metal ions, assuring that their activity will be low. Other complexing agents can also be used as long as they are not electrooxidized or electroreduced at the potential at which the electrode is poised.
Glucose oxidase is a flavoprotein enzyme that catalyzes the oxidation by dioxygen of D-glucose to D-glucono-1,5-lactone and hydrogen peroxide. Reduced transition metal cations such as, for example, Fe2+, and some transition metal complexes, can react with hydrogen peroxide. These reactions form destructive OH radicals and the corresponding oxidized cations. The presence of these newly formed transition metal cations can inhibit the enzyme and react with the metal complex. Also, the oxidized transition metal cation can be reduced by the FADH2 centers of an enzyme, or by the transition metal complex.
Inhibition of the active site of an enzyme or a transition metal complex by a transition metal cation, as well as damaging reactions with OH radicals can be alleviated, thus increasing the sensitivity and functionality of the electrodes by incorporating non-cyclic polyphosphates, as discussed above. Because the polyphosphate/metal cation complex typically has a high (oxidizing) redox potential, its rate of oxidation by hydrogen peroxide is usually slow. Alternatively, an enzyme such as, for example, catalase, can be employed to degrade hydrogen peroxide.